This is a proposal to continue studies of the mechanism of initiation and regulation of eukaryotic ribosomal RNA transcription. The organism being studied, Acanthamoeba castellanii, is an excellent model for the study of eukaryotic transcription using biochemical approaches, and significant findings during the last funding period lead logically into the proposed experiments. Specifically, it has been shown that DNA-dependent RNA polymerase I (pol I) binds to the promoter, without regard to the DNA sequence in the pol I binding site, by protein-protein interaction with transcription initiation factor (TIF), a protein which binds to a specific DNA sequence in the core promoter. Yet, some of these complexes with altered sequence under the bound pol I are unable to initiate transcription. Experiments are planned to determine why this occurs, with the initial hypothesis being a sequence effect on melting. Melting has been shown to be a separate step in the initiation process requiring the presence of nucleotide substrates. Is there an energy requirement for this step? Is it necessary to form phosphodiester bonds for stable melting to occur, or is nucleotide binding to the polymerase sufficient? Exactly when does melting occur, and exactly how many base pairs are melted? How dose DNA supercoiling affect melting and promoter efficiency? The role, if any, of the so called non-transcribed spacer (NTS) in promoting or enhancing rRNa transcription will be evaluated. Experiments include sequencing and footprinting of the NTS, analysis of NTS competition with the gene promoter and analysis of the effect of multiple TIF binding sites upstream of the promoter. The latter should also give an insight into the role of specific sites of transcription termination, and perhaps pol I enhancers. The mechanism of binding of TIF and pol I to promoter DNA will be detailed by carrying out premodification footprinting techniques. Questions to be addressed include: What is the binding constant for TIF, and for polymerase? Is the DNA bent by TIF? What are the phosphate contacts? What are the sugar contacts? Does TIF binding affect DNA conformation in the RNA polymerase binding region? As mentioned above, Acanthamoeba pol I binds to the rRNA promoter solely by protein-protein interactions with TIF. This protein-protein interaction will be analyzed by chemical and photochemical cross-linking studies. In order to fully carry out this analysis, pol I subunits will be cloned, and the effect of site-directed mutagenesis analyzed by novel techniques. This laboratory demonstrated that ribosomal RNA transcription is regulated in eukaryotic cells by modification of pol I. The effect of modification is to prevent pol I from binding to the promoter, and by inference, modification affects the interaction between TIF and the polymerase. Preliminary evidence shows a modification to occur on the 39KD subunit of pol I. Experiments to correlate this modification (or others found later) with regulation are planned. The chemical nature of the modification will be determined. The enzyme which carries out modification will be identified, and its mechanism of response to cellular growth rate investigated. In addition to its serving as a model system for studies of transcription mechanisms, Acanthamoeba is the causative agent of amoebic keratitis, a serious and currently difficult to treat corneal infection associated with contact lens wear, lending direct health relevance to this project.